The isolation and purification of microorganisms and their nucleic acids from biological materials represent a fundamental technique for molecular-biological analysis and diagnosis in human or veterinary medicine. Infections need to be detected quickly and reliably, to ensure that appropriate therapy is undertaken without delay.
Many biological samples and materials of interest contain substances that reduce the efficiency of nucleic acid testings. For example, it is well known that many substances that inhibit enzyme activity are present in many types of cells and can limit the use of amplification assays such as PCR. For example, heme, the oxygen carrier in blood, as well as its derivatives, can inhibit PCR amplification of target DNA in samples containing blood. Heme's breakdown products, such as bilirubin, as well as bile salts can inhibit PCR in samples containing feces. In addition, many of the reagents used to cultivate microorganisms or to prepare samples for PCR can inhibit amplification when present at contaminating levels including Triton, Sodium Dodecyl Sulfate (SDS) and others.
Furthermore, host cells (i.e., cells naturally present in the sample being tested for the presence of contaminating microbial cells e.g., a patient's red blood cells and white blood cells) in biological samples tested for the presence of microbial cells contribute to the “dilution” of microbial cells nucleic acids and leads to a decrease in sensitivity of the diagnostic assay. Hence, it would be advantageous to separate and remove as much host's cells as possible in the biological sample while maintaining the microbial cells integrity to increase the sensitivity of nucleic acid testing.
Attempts have been made to remove amplification inhibitors (e.g., host cells, etc.) introduced by conventional whole blood processing methods either by 1) isolating the nucleic acids from the sample prior to nucleic acid analysis; or 2) diluting the processed sample to reduce the effect of inhibitors. Some conventional protocols for nucleic acid analysis of whole blood rely on initial volumes of sample as small as 2-100 μl to reduce inhibitors to an acceptable level. Isolation of nucleic acids is cumbersome and requires that a high concentration of nucleic acid be present to be effective. Dilution or use of small sample volumes significantly compromises the sensitivity of the nucleic acid analysis.
For example, WO 2009/015484 describes a method for isolating microorganisms and/or microorganisms' nucleic acids from bodily fluids comprising treating the sample with a filtered and autoclaved Saponin solution at a concentration between 20 and 100 mg/ml. The method involves the separation of host cells from microbial cells prior to nucleic acid extraction.
EP 0 745 849 describes a method which eliminates inhibitors that interfere in particular with enzymatic nucleic acid reactions and which is also compatible with conventional culturing techniques. Selective lysis of red blood cells is achieved with Triton or Saponin at a final concentration between 0.1 and 0.2%, followed by centrifugation at 5,000-15,000×g for 5-30 min. and subsequent washings to remove inhibitors present in whole blood or introduced by reagents used in the sample processing protocol. The sample volume that may be processed using this method is up to 5 ml. Although this method constitutes an improvement over the previous methods it still requires multiple fold dilution of the initial volume of sample as well as high speed centrifugation. Hence the use of concentrating processing steps is required to obtain sufficiently concentrated nucleic acids for further analysis. In addition, Saponin is a chemically complex substance made of various chemical compounds and Applicants have observed that it is prone to lot-to-lot variability which reduces reproducibility. Moreover, at concentrations employed, Saponins do not lyse white blood cells such as macrophages, thereby contributing to the “dilution” of microbial cells which often results in a decrease in sensitivity.
Some commercially available products for purifying nucleic acids from bodily fluids involve the simultaneous lysis of red and white blood cells as well as microbial cells (SeptiFast™ prep kit from Roche Diagnostics; IsoQuick™ nucleic acid extraction kit from ISC BioExpress; and Nuclisens™ easyMAG™ system from Biomerieux). A disadvantage of this approach is the presence of a larger proportion of blood cells nucleic acids than of microbial nucleic acids which reduces sensitivity of microbial detection in the sample. In addition, systems like SeptiFast™ prep kit require numerous handling steps and take about two hours of treatment prior to extraction of human and microbial DNA. Furthermore it was shown that a majority of blood samples collected from septicemic patients may contain as low as 10 colony Forming Unit (CFU) of microbial cells/ml of blood (Johnson et al., 1993, APMIS, 101:595-601), which may be insufficient to allow detection using these processing methods. For example, the analytical sensitivity of the SeptiFast™ prep kit is approximately 30 CFU of microbe/nil of blood which is well over the concentration of microbe observed in some septicemic patient.
Accordingly, the ideal sample processing method for purification and isolation of microbial cells from biological samples and subsequent release of nucleic acids for nucleic acid testing would include the following features: 1) removes amplification and detection inhibitors, in particular those introduced by lysis of the hosts cells (e.g., red blood cells); 2) releases a sufficient amount of nucleic acids from the microorganisms for amplification; 3) enable the processing of large sample volumes to improve detection sensitivity; 4) uses a single protocol, that allows for the recovery of viable and intact microbial cells which can subsequently be cultured for biochemical testing; and 5) is simple, rapid and requires a limited number of processing steps to reduce possible cross contamination and the time before which a diagnostic is available.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.